Suppressing breast cancer by exercise: consideration to animal models and exercise protocols

[Purpose] Exercise is thought to have a significant effect on chemotherapy, and previous studies have reported that exercise can increase patient survival. Thus, in this review, we aimed to summarize various animal models to analyze the effects of exercise on breast cancer. [Methods] We summarized types of breast cancer animal models from various reports and analyzed the effects of exercise on anti-cancer factors in breast cancer animal models. [Results] This review aimed to systematically investigate if exercise could aid in suppressing breast cancer. Our study includes (a) increase in survival rate through exercise; (b) the intensity of exercise should be consistent and increased; (c) a mechanism for inhibiting carcinogenesis through exercise; (d) effects of exercise on anti-cancer function. [Conclusion] This review suggested the necessity of a variety of animal models for preclinical studies prior to breast cancer clinical trials. It also provides evidence to support the view that exercise plays an important role in the prevention or treatment of breast cancer by influencing anticancer factors.


INTRODUCTION
Non-communicable diseases, as chronic diseases, account for 70% of the mortality rates worldwide, while communicable diseases cause the remaining 30%. Among non-communicable diseases, those with high mortality rates include cancer, diabetes, cardiovascular disease, and lung disease. Cancer is classified as a fatal disease for patients because it is difficult to cure owing to its rapid growth, and is highly likely to spread throughout the body through blood or lymphatic fluid. In particular, reduction of female physical activity due to various social environments is a potential cause of increased breast cancer incidence. In addition, the most frequent characteristic of breast cancer among women is not only high incidence, but also high efficiency of cancer treatment. Although the effectiveness of anti-cancer drugs is very high for breast cancer patients, a lot of pain, caused by chemotherapy, accompanies it. Therefore, there is an urgent need to improve the survival rate of breast cancer patients, and improve the quality of life during chemotherapy treatment. Recently, various preclinical studies have suggested that exercise attenuates tumor growth and tumorigenesis (Table 1). However, molecular mechanisms by which exercise affects cancer progression are not yet clear. In this review, we aimed to summarize studies on exercise methods that could potentially increase the survival rate of breast cancer patients and suppress cancer progression. main ways of cancer treatments: 1) surgery, 2) chemotherapy, 3) radiation therapy, and 4) hormone therapy. Prophylactic surgery suppresses the cancer progression by performing a biopsy for the purpose of diagnosis through surgery or removing the benign tumor completely. Surgery also prevents the spread of cancer to other cells in the body and helps relieve symptoms. Chemotherapy refers to the use of therapeutic agents for regulating hyperproliferative cells. Radiation therapy kills cancer cells by directly irradiating them. Hormone therapy that suppresses estrogen action is also used as a cancer treatment method, as breast cancer is affected by estrogen levels, unlike other cancers.

Exercise regulates the breast cancer in animal models by inhibiting carcinogenesis
Disease increase over the last two decades may be due to a more westernized lifestyle, which is accompanied by excessive nutrition and lack of exercise 3 . Guidelines on cancer prevention are well known, and include recommendations for controlling metabolism, such as a balanced nutrient intake, eating vegetables, regulating vitamin intake, and controlling weight. Furthermore, exercise can prevent and treat various diseases, and in recent years, research on anti-cancer efficacy has been actively conducted.
Physical activities of Korean women are very low compared to women in other countries. Moreover, many women have adopted western food and a sedentary lifestyle, which has led to reduced voluntary exercise. The highest incidence of cancer among Korean women is breast cancer, and it has been suggested that breast cancer may be related to metabolic problems. Therefore, the effectiveness of exercise for the treatment or prevention of breast cancer should be investigated in future clinical studies.
An experimental laboratory animal is defined as an animal developed and improved for use in accordance with the purpose of test, diagnosis, education, research, and biological products in the research process. Among laboratory animals, primates such as Callithrix jacchus and Macaca fascicularis are most similar to humans; however, there exist issues regarding the ethics of conducting research using these animals. Rodents such as Mus muculus, Rattus norvegicus, and Cavia porcellus are the most commonly used experimental animals. In particular, Mus muculus has a genetic similarity with humans (approximately >80%), and a biologically similar body structure, a short pregnancy period (19 -21 days) is also advantageous for preclinical studies. Therefore, Mus muculus has been used as a knockout mouse, cancer model xenograft, orthotropic model, and chemically induced-disease model. To develop a mouse model of breast cancer, it is necessary to have experimental cells, and patient derived primary cells such as MCF-7 (ER+, PR+, HER2-), MDA-MD-231 (ER+, PR+, HER2-), MC4-L2 (ER+), E0771 (ER+) and 4T1 (ER-) (Fig. 1).
The breast cancer animal model consists of chemically induced models, transgenic mice models, orthotopic mice models, and xenograft models. In the case of chemically induced breast cancer models, 7,12-dimethylbenzanthracene (DMBA; 1 mg/mL weekly, for six weeks) is injected subcutaneously into the side of the abdomen. Poly-aromatic structure of lipophilic molecule, DMBA has high carcinogen activity in the breast. To evaluate tumor progression, mice are established with genetic modifications that target the oncogene, such as simian virus 40 (SV40) T antigens and polymer middle T antigen (PyMT). In the establishment of mice models by injection with breast cancer cells, mice are mainly used in the study of tumor biology and pharmacology, as these models retain the biological properties of cancer. Breast cancer cells are injected into the mammary fat pad of host mice to obtain orthotropic models. In this case, the number of cells used is appropriate (1 x 10 5 to 1 x 10 6 /mouse), and cancer cells injected into the mouse organs exhibit properties similar to breast cancer generated in the human body over time, and can be correlated to metastatic cancer. To develop a xenograft model, cells (1 x 10 6 to 1 x 10 7 /mouse) are injected subcutaneously into the dorsal side of the mouse.
Using these various animal models, studies on the ben-  Table 1). Tumors are defined as transformed cells that undergo abnormal or rapid proliferation, beyond normal regulatory functions, in the organism. Tumors are divided into two types: benign neoplasms and carcinomas. Benign neoplasms have a relatively slow growth rate, and do not penetrate or spread into other tissues. In contrast, carcinomas rapidly grow, and invade other tissues and metastasize. The process of tumor development by carcinogenesis is a multi-step process. The first stage is initiation, where normal cellular DNA is attacked by carcinogens, leading to genetic modification and irreversible mutations. The second stage is promotion, wherein cell proliferation is actively performed to maintain and promote the population of mutant cells to counter immune response in vivo as it eliminates abnormal cells. The third step is progression, the process of increasing the characteristics of a malignant tumor by converting it from a benign tumor to a malignant tumor. In the process of tumor development, the morphology and function of normal cells are altered by genetic modification through internal or external stimulants. External factors include chemical carcinogens such as smoking, physical stimuli such as radiation, and RNA tumor viruses such as HTLV-1 virus. Internal factors involved in the mutation of the target gene include oncogene and tumor suppressor genes. Tumor suppressor genes include TGF-β, E-Cadherin, NF-1, PTEN, SAMD2, SMAD4, and p53, and regulate cell population through apoptosis and proliferation. Oncogene mutation targets are cell cycle regulatory genes such as cyclin D1, Her2, and K-ras. Many preclinical studies suggest that the beneficial effect of exercise training in cancer progression is brought about by direct regulation of intertumoral factors, i.e., tumor growth rate, metastasis, and tumor immunogenicity (Table 1).   Taken together, these data suggest that the anti-cancer activity of the exercise protocols is involved in endurance and moderate-intensity exercise. If so, which mechanism of exercise showed an anti-cancer effect? Results strongly suggest that exercise inhibits epigenetic modification of tumor cells, but enhances apoptosis and immune suppression 29 . Reactive oxygen species (ROS) perform signal transduction in vivo; however, excessive production can cause oxidative stress, which leads to cancer 30 . Moderate intensity exercise can regulate ROS and biological signaling in vivo 31 . It is likely that exercise is related to the regulation of the reactive oxygen species (ROS)-involved microenvironment of cancer 32 . Therefore, these studies also suggest that controlling ROS a potential mechanism for the treatment of cancer 33 .
However, this claim raises further questions as to why exercise is closely related to change in the microenvironment of cancer. One possible belief is that exercise can exert anti-cancer effects by solving problems that arise during metabolic processes. During carcinogenesis, most tumor cells exert cell growth signaling pathway via glucose metabolic reprograming 34 . Recent study suggests that effective anti-cancer effect could be related to the regulation of metabolic syndrome 35 . The results supporting these claims are as follows: First, exercise can lead to activation of natural killer cell, lymphocyte, consequently resulting in the regulation of the tumor growth and metastasis 36 . In addition, exercise attenuates tumorigenesis and tumor progression 37 . Next, the ketone diet (KD) is characterized by high fat, adequate protein, and very low carbohydrate compositions. Some studies have reported that the physiological phenomena caused by exercise or fasting are very similar to physiological conditions observed in the KD 38 . Various preclinical studies have shown that exercise or the KD displays anti-cancer efficacy 39,40 . Taken together, a possible hypothesis is that exercise-binding KD modulates metabolic dysfunction and causes internal factors, which involved in the mutation of the target gene such as ROS generation and tumor-suppressor gene mutations, thereby suggesting its potential as a cancer therapeutic.
However, the anti-cancer effects of KD and exercise can be contradictory. Acute exercise did not change tumor formation, but continuous steady aerobic exercise displayed effective anticancer effects. The general view presented in many studies is that exercise exerts an anti-cancer effect by reducing the size of tumors, promoting energy metabolism, and increasing immune activity by constant exercise. Therefore, further studies should investigate that find and apply an appropriate energy source for exercise that show anticancer efficacy.

CONCLUSION
Various preclinical studies have shown that exercise weakens tumor growth and tumor development. Moreover, these studies suggest that mice bearing breast cancer exhibited anti-cancer effects by increasing immune responses and anti-inflammatory factor levels through acclimation of increased exercise intensity every week. Thus, continuous exercise can have potential medical benefits as a prevention or therapeutic method for breast cancer. To facilitate this research, researchers need to study the etiological mechanisms that rely on clinical features with underlying pathological features of the disease, as well as based on mechanisms not necessarily present in patients. For example, using animal models to discover new treatments for a variety of diseases is an essential element in discovering new therapeutic targets and performing drug testing at the preclinical stage.